The goals of this FIRCA application address fundamental cellular processes by exploring molecular mechanisms regulating cargo progression through the secretory pathway. The proposal builds on research interests of collaborating investigators at the University of Alabama at Birmingham and the University of Cordoba, Argentina. The proposal is an extension of a NIH grant RO1 GM62696-06 entitled "Role of p115 in Membrane Traffic". The parent grant addresses basic mechanisms of ER-Golgi traffic by analyzing the role of the transport factor p115. P115 functions in ER-Golgi traffic by participating in protein-proteins interaction with distinct proteins localized in different compartments along the pathway. The experimental goals of the parent grant are to explore p115 function at the Golgi by analyzing tethering interactions of p115 with two Golgi proteins, GM130 and giantin. We now wish to expand the scope of our inquiry by exploring p115-mediated events in pre-Golgi stages of traffic. This FIRCA proposal is based on our recent findings that p115 may act at the level of ER exit sites to generate pre-Golgi transport intermediates. We now plan to test the hypothesis that cargo and p115 are coordinately recruited at ER exit sites and that p115 association is required for the formation of vesicular tubular clusters (VTCs) that transport cargo and p115 to the Golgi. A series of experimental approaches including biochemical, molecular, cellular and imaging technologies will be used to address the following Specific Aims: 1) define how p115 associates with ER exit sites; 2) define how p115 traffics to the Golgi; and 3) define how p115 functions in pre-Golgi transport. Completion of proposed studies will extend our understanding of how p115 is recruited to membranes and moves along the linear secretory pathway, and will provide first ever inquiry into the molecular events by which p115 mediates the formation of transport competent VTCs.